Confocal scanning imaging systems are well-known in the art. Because of their ability to reject out-of-focus light and to image and reconstruct high resolution images of three-dimensional objects at all focal points on the object, they have been used in a wide variety of applications from industrial to biological technologies. High resolution is achieved by scanning the object in small increments at each x-y-z coordinate plane of focus.
The confocal imaging process is as follows. The confocal imaging system sequentially illuminates small focal regions of an object until the entire desired portion of the object is scanned. During the scanning process, reflected light from the small focal region is acquired. A user may view the image with a camera and display monitor. Additionally, the acquired image may be stored with an image processor and computer. A composite of all the small focal regions scanned results in a reconstructed image of the desired portion of the object itself with high resolution. Confocal imaging optically filters out light from regions outside the plane of focus at each small focal region to minimize scattered and reflected light interferences.
The use of apertures is essential to confocal imaging systems. The incident light illuminates only a small incremental focal plane or region on the object. In the return path, the aperture is located in a plane conjugate with the focal plane containing an image of the focal point on the object. Optical signals from out-of-focus points or regions are rejected because of the use of these apertures and the nature of the illumination. In other words, by strategically placing the aperture in a plane conjugate to the focal plane, only those return light emanating from the immediate vicinity or region of the illuminated focal point passes through the aperture and into the sensor for detection and processing.
Most confocal imaging systems utilize at least two apertures. One aperture is for the incident light from the light source to illuminate the incremental image point on the object. Another aperture is used for the return light from the incremental image point on the object. The apertures are of various shapes and sizes. The plate on which the apertures are located are either stationary or rotatable.
Various techniques are used to align or synchronize these two apertures as each incremental image point is obtained. Furthermore, optical designers and engineers constantly confront the problem of matching the size and shape of the incident and return light apertures. Confocal imaging systems employing two apertures are expensive, difficult to use, and need fine tuning and adjustments to obtain high resolution images. Merely changing the beam splicers and filters, as is often required in fluorescence applications, detrimentally affects the alignment of the imaging system.
The advent of single aperture confocal imaging systems appeared to eliminate some of the inherent problems associated with multiple aperture confocal imaging systems. After all, the use of one aperture for both the incident and return light seemed to eliminate alignment problems. However, image quality was not as superior as expected. One cause of this problem was the design of the size of the aperture. Although the aperture must be large enough to transmit all incident light from the light source, it should not be so large so as to accept all return light from the object and other scattered and undesired reflected light. Improper aperture sizes caused lower resolution images. Another cause of this problem was that the reflected light from the aperture itself is many orders of magnitude greater than the actual signal coming from the object, especially for fluorescence applications.
Other single aperture confocal imaging systems fail because of theft inability to shield crucial incident and return light paths from unwanted interference such as scattered and reflected light. Thus, image resolution is much lower than theoretically predicted.
One embodiment of the present invention minimizes these problems in a single aperture confocal imaging system by tilting the aperture plate, making the two major surfaces optically flat, coating the aperture plate with anti-reflection material, sizing the aperture so that it transmits all of the incident light while acting as a spatial filter for the return light, using a matching pre-conditioning aperture so that the light incident on the aperture plate is contained within the aperture of the aperture plate, or a combination of the above. Other devices such as filters, beam splitters, light stops, and lenses may also be utilized to reject, reduce, or redirect scattered and undesired reflected light.
In one embodiment, a laser is used as the light source. The advantage of using lasers is its use as an intense, monochromatic light source having a high degree of coherence. By scanning point by point with a laser beam, the temporal and spatial coherence is reduced at the field plane while remaining coherent in the aperture plane. Use of lasers in fluorescence applications is particularly useful. Embodiments of the present invention can generate clear, thin optical sections of fluorescence imaging system images without interference from out-of-focus fluorescence.